Anti-human CD235a rabbit monoclonal antibody as well as preparation method and application thereof

Abstract

The invention relates to the technical field of immune globulin, in particular to an anti-human CD235a rabbit monoclonal antibody as well as a preparation method and application thereof. The amino acid sequences of CDR1, CDR2 and CDR3 of a light chain variable region of the anti-human CD235a rabbit monoclonal antibody provided by the invention are respectively as shown in SEQ ID NO.2-4; and the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region are as shown in SEQ ID NO.5-7. The anti-human CD235a rabbit monoclonal antibody provided by the invention can be specifically combined with CD235a protein, so that the specificity, sensitivity, accuracy and reliability of CD235a protein immunodetection are remarkably improved, and the anti-human CD235a rabbit monoclonal antibody can be suitable for marking erythrocyte lineage cells.

Description

Technical Field

[0001] This application relates to the field of immunoglobulin technology, specifically to an anti-human CD235a rabbit monoclonal antibody, its preparation method, and its application. Background Technology

[0002] CD235a, also known as Glycophorin A (blood group glycoprotein A), is encoded by the GYPA gene on chromosome 4q31.21 and is expressed throughout the entire process of erythropoiesis. High expression indicates erythrocyte maturation. Its high cell type specificity makes it an ideal marker for identifying erythroid cells. CD235a is a type I transmembrane salivary glycoprotein with a molecular weight of 39 kDa. Its extracellular region is rich in sialic acid (negatively charged), which prevents erythrocytes from aggregating or adhering to vascular endothelial cells in the bloodstream.

[0003] In most erythroleukemias, neoplastic nucleated erythrocytes express CD235a protein, while acute myeloid leukemia and acute lymphoblastic leukemia express almost no CD235a. Therefore, CD235a is mainly used for the auxiliary diagnosis of erythroleukemia and the identification of erythroid leukemia. In pathological biopsies, staining with anti-CD235a antibodies can be used to identify whether a tumor originates from the erythroid lineage (such as erythroleukemia) or to differentiate certain rare tumors. In scientific research, CD235a antibodies can be used to isolate purified erythroid precursor cells from bone marrow or peripheral blood using flow cytometry for studying erythropoiesis, hemoglobinopathies (such as thalassemia), and the mechanisms of malaria infection.

[0004] Therefore, developing highly specific anti-CD235a monoclonal antibodies to ensure that the antibodies bind only to CD235a and avoid cross-reaction with other proteins can reduce false positive or false negative results and improve diagnostic accuracy. With the help of CD235a monoclonal antibodies, the specific role of CD235a in the physiological and pathological processes of erythrocytes, as well as its interaction with other molecules, can be more clearly revealed. Summary of the Invention

[0005] In view of this, this application provides an anti-human CD235a rabbit monoclonal antibody, its preparation method and application. The anti-human CD235a rabbit monoclonal antibody provided by this application significantly improves the specificity and sensitivity of CD235a protein immunoassay and is suitable for labeling CD235a protein in erythroid cells.

[0006] In a first aspect, this application provides an anti-human CD235a rabbit monoclonal antibody or its antigen-binding fragment, comprising a light chain variable region and a heavy chain variable region. The light chain variable region includes complementarity-determining regions LCDR1, LCDR2, and LCDR3. The amino acid sequence of LCDR1 is shown in SEQ ID NO.2, the amino acid sequence of LCDR2 is YTS, and the amino acid sequence of LCDR3 is shown in SEQ ID NO.4. The heavy chain variable region includes complementarity-determining regions HCDR1, HCDR2, and HCDR3. The amino acid sequence of HCDR1 is shown in SEQ ID NO.5, the amino acid sequence of HCDR2 is shown in SEQ ID NO.6, and the amino acid sequence of HCDR3 is shown in SEQ ID NO.7.

[0007] Further, the light chain variable region includes an amino acid sequence having at least 95% identity with the amino acid sequence shown in SEQ ID NO:8; the heavy chain variable region includes an amino acid sequence having at least 95% identity with the amino acid sequence shown in SEQ ID NO:9; wherein, preferably, the amino acid sequence has 95%, 96%, 97%, 98% or 99% identity.

[0008] Furthermore, the light chain variable region includes the amino acid sequence shown in SEQ ID NO:8, and the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO:9.

[0009] In this application, the light chain variable region has a total length of 103 amino acids. The number of amino acids in the four domains of its FR are 26, 17, 36 and 10, respectively. The number of amino acids in the three domains of LCDR are 6, 3 and 5, respectively. The regions of LCDR1, LCDR2 and LCDR3 are 27aa-32aa, 50aa-52aa and 89-93aa, respectively. Their amino acid sequences are QSVYKM (SEQ ID NO.2), YTS (SEQ ID NO.3) and QAYSD (SEQ ID NO.4), respectively.

[0010] In this application, the total length of the heavy chain variable region is 107 amino acids. The number of amino acids in the four domains of its FR are 25, 16, 38 and 11, respectively. The number of amino acids in the three domains of HCDR are 6, 6 and 5, respectively. HCDR1, HCDR2 and HCDR3 are 26aa-31aa, 48aa-53aa and 92aa-96aa, respectively. Their amino acid sequences are: GIDLSN (SEQ ID NO.5), ISTGGT (SEQ ID NO.6) and ARRWG (SEQ ID NO.7), respectively.

[0011] Furthermore, the antigen-binding fragment is one of F(ab')2, Fab', Fab, Fv, scFv, dsFv, and bispecific antibodies.

[0012] Secondly, this application also provides a biomaterial, which is a polynucleotide, a carrier, or a cell. 1) The polynucleotide encodes the anti-human CD235a rabbit monoclonal antibody or its antigen-binding fragment as described in the first aspect; or 2) The vector carries the polynucleotide; or 3) The cell carries the polynucleotide, or contains the vector, or is able to express the anti-human CD235a rabbit monoclonal antibody or its antigen-binding fragment as described in the first aspect.

[0013] Thirdly, this application also provides a method for preparing the anti-human CD235a rabbit monoclonal antibody or its antigen-binding fragment as described in the first aspect, including culturing the cells described in the second aspect; The cells are prepared by converting cells with a polynucleotide encoding a fragment of the anti-human CD235a rabbit monoclonal antibody or its antigen-binding fragment. The polynucleotide includes a heavy chain expression plasmid and a light chain expression plasmid. The conversion includes co-converting the heavy chain expression plasmid and the light chain expression plasmid into the cells.

[0014] Furthermore, the cells are eukaryotic cells, preferably mammalian cells, more preferably 293 cells or CHO cells.

[0015] Fourthly, this application also provides the use of the anti-human CD235a rabbit monoclonal antibody or its antigen-binding fragment described in the first aspect, or the biological material described in the second aspect, in any of the following: 1) Used for purifying human CD235a protein; 2) Prepare a product for purifying human CD235a protein; 3) Detection of human CD235a protein for non-diagnostic and non-therapeutic purposes; 4) Prepare an immunoassay tool for detecting human CD235a protein; 5) Detection of erythrocyte lineage cells for non-diagnostic and non-therapeutic purposes; 6) Prepare immunoassay tools for detecting erythrocyte lineage cells; The immune detection tool is a detection reagent, reagent kit, test strip, or detection chip.

[0016] Fifthly, this application also provides an immunoassay tool for human CD235a protein, the immunoassay tool comprising the anti-human CD235a rabbit monoclonal antibody or its antigen-binding fragment as described in the first aspect, or the biological material as described in the second aspect.

[0017] Furthermore, the immunoassay tool is a test reagent, kit, test strip, or test chip, and is used for enzyme-linked immunosorbent assay (ELISA), Western blot, immunohistochemical staining (IHC), flow cytometry (FCM), and immunoprecipitation, etc.

[0018] Furthermore, the kit also includes antigen retrieval solution, endogenous peroxidase inhibitor (hydrogen peroxide), ultrasensitive enzyme-labeled goat anti-mouse / rabbit IgG polymer, DAB substrate buffer, DAB concentrated chromogenic solution, and hematoxylin staining solution.

[0019] Compared with existing technologies, the anti-human CD235a protein monoclonal antibody provided in this application can specifically bind to human CD235a protein, significantly improving the specificity, accuracy, and reliability of CD235a protein immunoassay. Furthermore, using the anti-human CD235a protein monoclonal antibody and kit of this application, IHC assay can clearly detect CD235a protein expression in erythroid cells, indicating that the anti-human CD235a protein monoclonal antibody of this application can be well applied to the immunohistochemical detection of CD235a protein in tissue cells. Attached Figure Description

[0020] To more clearly illustrate the specific embodiments of this application, the accompanying drawings used in the description of the specific embodiments will be briefly introduced below.

[0021] Figure 1 The image shows the results of the immunohistochemical detection of CD235a protein expression in nine normal tissues (lung, tonsils, colon, placenta, etc.) as described in Example 5. The primary antibody used was the anti-human CD235a rabbit monoclonal antibody OTIR2F6 provided in this application.

[0022] Figure 2 The image shows the results of the immunohistochemical detection of CD235a protein expression in four types of tumor tissues (lung cancer, papillary thyroid carcinoma, leiomyoma, and diffuse large B-cell lymphoma) as described in Example 5. The primary antibody used was the anti-human CD235a rabbit monoclonal antibody OTIR2F6 provided in this application. Detailed Implementation

[0023] The present application is further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the application. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions, conditions described in a laboratory manual, or conditions recommended by the manufacturer. Unless otherwise specified, the test reagents used in the following embodiments are conventional biochemical reagents; and the experimental methods described are conventional methods unless otherwise specified.

[0024] Example 1: Preparation of anti-human CD235a rabbit monoclonal antibody 1. Preparation of immunogens The characteristics of the CD235a protein sequence (GeneBank: NM_002099.8) were analyzed. Based on the hydrophilicity, three-dimensional structural features of CD235a, and the homology between human and rabbit sequences, amino acids 130-150 (SEQ ID NO.1) of the synthesized CD235a protein were selected as the detection material for magnetic bead screening and ELISA screening. After conjugation with KLH, it was used as an immunogen. The immunogen sequence was synthesized by Zhongtai Biochemical Co., Ltd.

[0025] 2. Animal immunization The CD235a protein immunogen obtained in step 1 was emulsified with Freund's adjuvant and administered subcutaneously to New Zealand white rabbits at a dose of 800 μg / rabbit. A second immunization was performed two weeks later, using incomplete Freund's adjuvant emulsification at a dose of 400 μg / rabbit. After three immunizations, blood was collected from the ear vein and serum titers were determined using serial dilutions with ELISA and IHC. The criteria for determining whether an immune response was generated in the immunized animals and whether the titer reached the level required for PBMC collection were based on the ELISA titer of 128,000 and the intensity of the staining signal in lung and placental tissues detected by IHC.

[0026] 3. PBMCs isolation, specific B cell sorting, and clonal recombination Heart blood was collected from New Zealand white rabbits whose serum titers met the standard. A 50mL syringe needle was inserted into the animal's heart to obtain the required blood volume. The blood collection was then immediately stopped, and the whole blood was transferred to a sterile 50mL tube. Following the instructions for use of the lymphocyte separation reagent, centrifugation was performed. The solution separated into four layers from top to bottom: a yellow plasma layer, a white thin film layer (i.e., a mononuclear cell layer), a separation solution layer, and a red blood cell layer. The mononuclear cell layer was aspirated and washed with PBS to remove platelets and lymphocyte separation solution, yielding rabbit PBMCs.

[0027] The CD235a naked peptide synthesized in step 1 was coupled with magnetic beads, and antigen-specific B cells were sorted and cultured. The synthesized CD235a naked peptide was then coated onto the B cell culture supernatant, and positive clones recognizing CD235a protein were screened by ELISA and IHC. The optimal clone was obtained after screening as OTIR2F6.

[0028] Positive clones were lysed with lysis buffer and the lysis buffer was collected. RNA was extracted from the lysate and then reverse transcribed into cDNA. The full-length light and heavy chain sequences of naturally paired rabbit monoclonal antibodies were amplified from the cDNA of the corresponding positive clones. Rabbit monoclonal antibody expression vectors were constructed using clonal recombination methods.

[0029] 4. Preparation and purification of monoclonal antibodies The heavy and light chain genes of the rabbit monoclonal antibody OTIR2F6 were loaded into an expression vector, and the recombinant plasmid was transfected into HEK293 cells. After 120-144 hours of transfection, the culture supernatant contained the recombinant rabbit monoclonal antibody recognizing human CD235a protein. The cell suspension was collected, the supernatant was obtained by centrifugation, and the antibody was purified by affinity chromatography. This yielded a rabbit monoclonal antibody specifically recognizing human CD235a protein. The concentration of the purified monoclonal antibody was determined by the BCA method, and the antibody specificity was then detected by IHC.

[0030] Example 2: Variable region gene and amino acid sequence analysis of anti-human CD235a monoclonal antibody Using the recombinant plasmid of the CD235a monoclonal antibody OTIR2F6 as a DNA template, the variable region light and heavy chain gene sequences of the antibody were amplified using 5' RACE (Rapid Amplification of cDNA Ends) technology. For detailed experimental procedures, please refer to the Takara Bio USA SMARTer® RACE 5' / 3' Kit user manual.

[0031] Based on the described antibody subtypes, specific gene primers pRace-H-GSP and pRace-K-GSP targeting the 3' end of their Ig and Kappa constant regions were designed. The primer sequences are as follows: pRace-H-GSP: 5'-CATCDGTCTATCCACTGGCCCCTG-3' (SEQ ID NO: 10); pRace-K-GSP: 5'-CTTCCCACCATCCAGTGAGCAGTT-3' (SEQ ID NO: 11).

[0032] DNA fragments of the heavy and light chains of the anti-human CD235a protein monoclonal antibody were amplified by RACE. The amplified light and heavy chains were ligated into the cloning vector PUC119 by enzyme digestion. Positive clones were selected by blue-white screening, and the positive plasmids were purified and sequenced using an ABI 3730 sequencer with universal primers M13F and M13R.

[0033] M13F: 5'-TGTAAAACGAGCGGCCAGT-3' (SEQ ID NO: 12); M13R: 5'-CAGGAAACAGCTATGAC-3' (SEQ ID NO: 13).

[0034] Data analysis was performed on the sequencing results of the nucleotide sequences of the light and heavy chains of the anti-human CD235a protein monoclonal antibody, and the amino acid sequence of the variable region of the light chain of the anti-human CD235a monoclonal antibody was shown in SEQ ID NO.8, and the amino acid sequence of the variable region of the heavy chain was shown in SEQ ID NO.9.

[0035] The light chain variable region is 103 amino acids in length. The number of amino acids in the four domains of its FR are 26, 17, 36 and 10, respectively. The number of amino acids in the three domains of LCDR are 6, 3 and 5, respectively. The regions of LCDR1, LCDR2 and LCDR3 are 27aa-32aa, 50aa-52aa and 89-93aa, respectively. Their amino acid sequences are QSVYKM (SEQ ID NO.2), YTS (SEQ ID NO.3) and QAYSD (SEQ ID NO.4), respectively.

[0036] The heavy chain variable region is 107 amino acids in length. The number of amino acids in the four domains of its FR are 25, 16, 38 and 11, respectively. The number of amino acids in the three domains of HCDR are 6, 6 and 5, respectively. HCDR1, HCDR2 and HCDR3 are 26aa-31aa, 48aa-53aa and 92aa-96aa, respectively. Their amino acid sequences are: GIDLSN (SEQ ID NO.5), ISTGGT (SEQ ID NO.6) and ARRWG (SEQ ID NO.7).

[0037] Example 3: Affinity Identification of Anti-human CD235a Monoclonal Antibody The non-competitive ELISA method for determining the antibody affinity constant (Ka) is as follows: 1) Coating: The synthesized naked CD235a peptide was diluted with carbonate buffer and the concentration gradient was set as follows: 2.5 μg / mL, 1.25 μg / mL, 0.625 μg / mL, 0.313 μg / mL. The solution was added to the coated plate in 100 μL wells and incubated overnight at 4°C.

[0038] 2) Blocking: After removing the coating solution, wash 3 times with PBS buffer, then incubate with 200 μL / well blocking buffer (5% skim milk powder) at 37°C for 1 h.

[0039] 3) Incubation of primary antibody: Serially dilute CD235a monoclonal antibody, with concentration gradients set as follows: 3.35 × 10⁻⁶ -2 μg / mL, 1.34×10 -2 μg / mL, 5.36×10 -3 μg / mL, 2.14×10 -3 μg / mL, 8.58×10 -4 μg / mL, 3.43×10-4 μg / mL, 1.37×10 -4 μg / mL was added to the coated plate at a rate of 100 μL / well and incubated at 37°C for 1 h.

[0040] 4) Incubation with secondary antibody: Remove primary antibody, wash 3 times with PBS buffer, dilute HRP-labeled goat anti-rabbit secondary antibody 1:20000, add 100 μL / well to the coated plate, incubate at 37℃ for 30 min.

[0041] 5) Color development and termination: Remove the secondary antibody, wash 3 times with PBS buffer, add 50 μL of color development solution to the coated plate at 37°C for 15 min, and then add 50 μL of stop solution (1 mol / L H2SO4) to terminate the reaction.

[0042] 6) Data reading: Set the parameter to 450nm in the microplate reader and read the absorbance data.

[0043] 7) Data Processing: An S-shaped curve was plotted with the logarithm of antibody concentration (mol / L) on the x-axis and the corresponding absorbance on the y-axis. The affinity constant was calculated, and the calculated affinity constant for the anti-human CD235a monoclonal antibody was Ka = 2.45 × 10⁻⁶. 10 L / mol.

[0044] Example 4: Immunohistochemical detection kit containing anti-human CD235a monoclonal antibody An immunohistochemical assay kit containing an anti-human CD235a protein monoclonal antibody includes antigen retrieval solution [1 mM EDTA, 10 mM Tris buffer (pH 8.0)], and is prepared as follows: in Example 1, the purified anti-human CD235a protein monoclonal antibody (0.134 μg / mL), endogenous peroxidase inhibitor (hydrogen peroxide), ultrasensitive enzyme-labeled goat anti-mouse / rabbit IgG polymer, DAB substrate buffer, DAB concentrated chromogenic solution, and hematoxylin staining solution are prepared.

[0045] Example 5: Detection of CD235a protein expression in different tissues using an anti-human CD235a immunohistochemical detection kit. The immunohistochemical detection kit described in Example 4 was used to detect CD235a protein in various tissues. The specific steps are as follows.

[0046] (1) The immunohistochemistry of this embodiment was used to select multiple tissues such as lung and placenta for verification. The tissue was sectioned using a Leica tissue slicer and the section thickness was set to 4μm.

[0047] (2) Dewaxing and hydration: The slices treated above were sequentially subjected to analytical grade xylene for 10 min 3 times, anhydrous ethanol for 1 min 3 times, 95% ethanol for 1 min, 85% ethanol for 1 min, 75% ethanol for 1 min, and deionized water for 2 min 3 times to achieve dewaxing and hydration.

[0048] (3) Antigen repair: Antigen repair is carried out using high temperature and high pressure. The repair conditions are: antigen repair solution (EDTA antigen repair solution, pH 8.0), heat repair for 2.5 min, natural cooling, and soaking in deionized water for 2 min 3 times.

[0049] (4) Inactivation: Use 3% hydrogen peroxide to inactivate endogenous peroxidase in tissues by standing at room temperature in the dark for 15 minutes, and then soak in deionized water for 2 minutes 3 times.

[0050] (5) Incubation with primary antibody: Add anti-human CD235a monoclonal antibody (0.134 μg / mL), place the slide in a humidified chamber, and incubate at 37°C for 60 min. Wash three times with 0.1% PBST for 2 min each time.

[0051] (6) Incubation with secondary antibody: Add 100 μL of high-sensitivity HRP-labeled goat anti-mouse / rabbit IgG polymer to the reaction area of ​​the slide and incubate at 37°C for 30 min. Wash three times with 0.1% PBST for 2 min each time.

[0052] (7) DAB color development: Add 120 μL of freshly prepared DAB color development solution, react at room temperature for 5 min, then stop the color development and wash 3 times.

[0053] (8) Hematoxylin counterstaining, differentiation and sapphire: stain in hematoxylin solution for 12s, wash 3 times to stop the color development, differentiate in 1% hydrochloric acid ethanol solution, rinse 3 times with deionized water to stop the reaction, put in Tris-EDTA disodium solution at pH 8.0 for sapphire, and rinse 3 times with deionized water.

[0054] (9) Dehydration and clearing: After blueing, the slides were treated with 75% ethanol for 1 min, 85% ethanol for 1 min, 95% ethanol for 1 min, 100% ethanol for 1 min 3 times, and xylene for 1 min 3 times, and then mounted with neutral resin.

[0055] (10) Microscopic examination: Observe the staining results under a microscope.

[0056] (11) Microscopic examination, such as Figures 1-2 As shown.

[0057] The expression status of CD235a protein in various normal and tumor tissues was detected by IHC assay using an anti-human CD235a protein monoclonal antibody.

[0058] Figure 1 Nine different tissues were shown, namely lung, liver, pancreas, placenta, tonsils, spleen, thyroid, colon, and appendix. The results showed that the brown-positive cells were all erythroid cells, while the other types of tissue cells did not show brown staining, that is, they did not express CD235a protein.

[0059] Figure 2 For example, tumors from different sources, namely lung cancer, papillary thyroid carcinoma, diffuse large B-cell lymphoma, and leiomyoma, showed that tumor cells, lymphocytes, alveolar epithelial cells, etc., did not have positive staining, and the brown positive staining was erythrocytes.

[0060] The above results are consistent with the theoretical expression pattern of CD235a. The concentration of the monoclonal antibody used for the detection was 0.134 μg / mL. The staining results under these conditions meet the clinical interpretation requirements and do not produce background staining that would affect diagnosis. Therefore, it can be demonstrated that the anti-human CD235a rabbit monoclonal antibody OTIR2F6 provided in this application has good specificity and high sensitivity.

[0061] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A rabbit monoclonal antibody against human CD235a or its antigen-binding fragment, characterized in that, It includes a light chain variable region and a heavy chain variable region. The light chain variable region includes complementarity-determining regions LCDR1, LCDR2, and LCDR3. The amino acid sequence of LCDR1 is shown in SEQ ID NO.2, the amino acid sequence of LCDR2 is YTS, and the amino acid sequence of LCDR3 is shown in SEQ ID NO.

4. The heavy chain variable region includes complementarity-determining regions HCDR1, HCDR2, and HCDR3. The amino acid sequence of HCDR1 is shown in SEQ ID NO.5, the amino acid sequence of HCDR2 is shown in SEQ ID NO.6, and the amino acid sequence of HCDR3 is shown in SEQ ID NO.

7.

2. The anti-human CD235a rabbit monoclonal antibody or its antigen-binding fragment according to claim 1, characterized in that, The light chain variable region includes an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO:8; the heavy chain variable region includes an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO:

9.

3. The anti-human CD235a rabbit monoclonal antibody or its antigen-binding fragment according to claim 2, characterized in that, The light chain variable region includes the amino acid sequence shown in SEQ ID NO:8, and the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO:

9.

4. The rabbit monoclonal antibody mAb6 or its antigen-binding fragment according to claim 1 or 2, characterized in that, The antigen-binding fragment is one of F(ab')2, Fab', Fab, Fv, scFv, dsFv, and bispecific antibodies.

5. A biomaterial, characterized in that, The biological material is a polynucleotide, a carrier, or a cell; 1) the polynucleotide encodes the anti-human CD235a rabbit monoclonal antibody or its antigen-binding fragment as described in any one of claims 1-4; or 2) The vector carries the polynucleotide; or 3) The cell carries the polynucleotide, or contains the vector, or is able to express the anti-human CD235a rabbit monoclonal antibody or its antigen-binding fragment as described in any one of claims 1-4.

6. The method for preparing the anti-human CD235a rabbit monoclonal antibody or its antigen-binding fragment according to any one of claims 1-4, characterized in that, Includes culturing the cells as described in claim 5; The cells are prepared by converting cells with a polynucleotide encoding a fragment of the anti-human CD235a rabbit monoclonal antibody or its antigen-binding fragment. The polynucleotide includes a heavy chain expression plasmid and a light chain expression plasmid. The conversion includes co-converting the heavy chain expression plasmid and the light chain expression plasmid into the cells.

7. The preparation method according to claim 6, characterized in that, The cells are eukaryotic cells, preferably mammalian cells, and more preferably 293 cells or CHO cells.

8. The use of the anti-human CD235a rabbit monoclonal antibody or its antigen-binding fragment according to any one of claims 1-4, or the biological material according to claim 5, in any of the following: 1) Used for purifying human CD235a protein; 2) Prepare a product for purifying human CD235a protein; 3) Detection of human CD235a protein for non-diagnostic and non-therapeutic purposes; 4) Prepare an immunoassay tool for detecting human CD235a protein; 5) Detection of erythrocyte lineage cells for non-diagnostic and non-therapeutic purposes; 6) Prepare immunoassay tools for detecting erythrocyte lineage cells; The immune detection tool is a detection reagent, reagent kit, test strip, or detection chip.

9. An immunoassay tool for human CD235a protein, characterized in that, The immunoassay tool comprises the anti-human CD235a rabbit monoclonal antibody or its antigen-binding fragment as described in any one of claims 1-4, or the biological material as described in claim 5.

10. The immunoassay tool according to claim 9, characterized in that, The immunoassay tool is a test reagent, kit, test strip, or test chip. The immunoassay tool is used for enzyme-linked immunosorbent assay (ELISA), Western blot, immunohistochemical staining (IHC), flow cytometry (FCM), and immunoprecipitation, etc.

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